Method of targeting specific cell populations using cell-binding agent maytansinoid conjugates linked via a non-cleavable linker, said conjugates and methods of making said conjugates

ABSTRACT

The present invention discloses a method for targeting maytansinoids to a selected cell population, the method comprising contacting a cell population or tissue suspected of containing the selected cell population with a cell-binding agent maytansinoid conjugate, wherein one or more maytansinoids is covalently linked to the cell-binding agent via a non-cleavable linker and the cell-binding agent binds to cells of the selected cell population.

This application is a continuation of U.S. application Ser. No.11/927,235, filed Oct. 29, 2007, which is a divisional of U.S.application Ser. No. 10/960,602, filed Oct. 8, 2004, which claimsbenefit of Provisional U.S. Patent Application No. 60/509,901, filedOct. 10, 2003, the entire disclosures of which are incorporated byreference.

FIELD OF THE INVENTION

A method consistent with the present invention relates to targetingspecific cell populations using cell-binding agent maytansinoidconjugates linked via a non-cleavable linker. Another method consistentwith the present invention is a method of making the conjugate. Acomposition consistent with the present invention relates to novelcell-binding agent maytansinoid conjugates where the maytansinoid islinked via a non-cleavable linker to the cell-binding agent. Anothercomposition consistent with the present invention relates to novelmaytansinoid esters.

BACKGROUND OF THE INVENTION

Maytansinoids are highly cytotoxic drugs. Maytansine was first isolatedby Kupchan et al. from the east African shrub Maytenus serrata and shownto be 100- to 1000-fold more cytotoxic than conventional cancerchemotherapeutic agents like methotrexate, daunorubicin, and vincristine(U.S. Pat. No. 3,896,111). Subsequently, it was discovered that somemicrobes also produce maytansinoids, such as maytansinol and C-3 estersof maytansinol (U.S. Pat. No. 4,151,042). Synthetic C-3 esters ofmaytansinol and analogues of maytansinol have also been reported(Kupchan et al., 21 J. Med. Chem. 31-37 (1978); Higashide et al. 270Nature 721-722 (1977); Kawai et al., 32 Chem. Pharm. Bull. 3441-3451(1984)). Examples of analogues of maytansinol from which C-3 esters havebeen prepared include maytansinol with modifications on the aromaticring (e.g. dechloro) or at the C-9, C-14 (e.g. hydroxylated methylgroup), C-15, C-18, C-20 and C-4,5.

The naturally occurring and synthetic C-3 esters can be classified intotwo groups:

-   (a) C-3 esters with simple carboxylic acids (U.S. Pat. Nos.    4,248,870; 4,265,814; 4,308,268; 4,308,269; 4,309,428; 4,317,821;    4,322,348; and 4,331,598), and-   (b) C-3 esters with derivatives of N-methyl-L-alanine (U.S. Pat.    Nos. 4,137,230 and 4,260,608; and Kawai et al., 32 Chem. Pharm.    Bull. 3441-3451 (1984)).

Esters of group (b) were found to be much more cytotoxic than esters ofgroup (a).

Maytansine is a mitotic inhibitor. Treatment of L1210 cells in vivo withmaytansine has been reported to result in 67% of the cells accumulatingin mitosis. Untreated control cells were reported to demonstrate amitotic index ranging from between 3.2 to 5.8% (Sieber et al., 43 Bibl.Haematol. 495-500 (1976)). Experiments with sea urchin eggs and clameggs have suggested that maytansine inhibits mitosis by interfering withthe formation of microtubules through the inhibition of thepolymerization of the microtubule protein, tubulin (Remillard et al.,189 Science 1002-1005 (1975)).

In vitro, P388, L1210, and LY5178 murine leukemic cell suspensions havebeen found to be inhibited by maytansine at doses of 10⁻³ to 10⁻¹ μg/mlwith the P388 line being the most sensitive. Maytansine has also beenshown to be an active inhibitor of in vitro growth of humannasopharyngeal carcinoma cells, and the human acute lymphoblasticleukemia line C.E.M. was reported inhibited by concentrations as low as10⁻⁷ μg/ml (Wolpert-DeFillippes et al., 24 Biochem. Pharmacol. 1735-1738(1975)).

Maytansine has also been shown to be active in vivo. Tumor growth in theP388 lymphocytic leukemia system was shown to be inhibited over a 50- to100-fold dosage range, which suggested a high therapeutic index; alsosignificant inhibitory activity could be demonstrated with the L1210mouse leukemia system, the human Lewis lung carcinoma system and thehuman B-16 melanocarcinoma system (Kupchan, 33 Ped. Proc 2288-2295(1974)).

Because the maytansinoids are highly cytotoxic, they were expected to beof use in the treatment of many diseases such as cancer. Thisexpectation has yet to be realized. Clinical trials with maytansine werenot favorable due to a number of side effects (Issel et al., 5 CancerTreat. Rev. 199-207 (1978)). Adverse effects to the central nervoussystem and gastrointestinal symptoms were responsible for some patientsrefusing further therapy (Issel at 204), and it appeared that maytansinewas associated with peripheral neuropathy that might be cumulative(Issel at 207).

Accordingly, targeting techniques to selectively deliver drugs to thetarget cell were employed. Both cleavable and non-cleavable linkers havebeen investigated for several drugs, but in most cases, including thecase of maytansinoids, in vitro cytotoxicity tests have revealed thatantibody-drug conjugates rarely achieve the same cytotoxic potency asthe free unconjugated drugs. Thus, it has been generally accepted thatfor targeted delivery of maytansinoids to be effective, the linkagebetween the maytansinoid and the cell-binding agent must be cleavable.

Furthermore, in the area of immunotoxins, conjugates containing linkerswith disulfide bridges between monoclonal antibodies and catalyticallyactive protein toxins were shown to be more cytotoxic than conjugatescontaining other linkers. See, Lambert et al., 260 J. Biol. Chem.12035-12041 (1985); Lambert et al., in Immunotoxins 175-209 (A. Frankel,ed. 1988), and Ghetie et al., 48 Cancer Res. 2610-2617 (1988). This wasattributed to the high intracellular concentration of glutathionecontributing to the efficient cleavage of the disulfide bond between anantibody molecule and a toxin. More recently, a conjugate ofmaytansinoids linked to the anti-Her2 breast cancer antibody TA.1 viathe non-cleavable linker SMCC was shown to be 200-fold less potent thana conjugate of maytansinoids linked to TA.1 via a linker having acleavable disulfide bond (Chari et al., 52 Cancer Res. 127-133 (1992)).

Thus, cytotoxic conjugates linked via disulfide-containing cleavablelinkers have been sought. Shen et al. described the conversion ofmethotrexate into a mercaptoethylamide derivative followed byconjugation with poly-D-lysine via a disulfide bond (260 J. Biol. Chem.10905-10908 (1985)). Preparation of a conjugate of thetrisulfide-containing toxic drug calicheamycin with an antibody was alsodescribed (Menendez et al., Fourth International Conference onMonoclonal Antibody Immunoconjugates for Cancer, San Diego, Abstract 81(1989)).

U.S. Pat. Nos. 5,208,020 and 5,416,064, the entire disclosures of whichare expressly incorporated herein by reference, disclose cytotoxicconjugates comprising cell-binding agents linked to specificmaytansinoid derivatives via cleavable linkers, such as linkerscontaining disulfide groups, linkers containing acid-labile groups,linkers containing photo-labile groups, linkers containingpeptidase-labile groups, and linkers containing esterase-labile groups

U.S. Pat. No. 6,333,410 B1, the entire disclosure of which is expresslyincorporated herein by reference, discloses a process for preparing andpurifying thiol-containing maytansinoids for linking to cell-bindingagents, and U.S. Pat. No. 6,441,163 B1, the entire disclosure of whichis expressly incorporated herein by reference, discloses a one-stepmethod for preparing cytotoxic conjugates of maytansinoids andcell-binding agents, wherein the linker is a disulfide-containingcleavable linker.

Furthermore, U.S. Pat. No. 5,208,020 teaches antibody-maytansinoidconjugates with non-cleavable linkers, wherein the linker comprises amaleimido group. However, the reference contains no experimental datademonstrating that such conjugates are effective to treat disease.

It has now been found, unexpectedly, that cytotoxic conjugates ofmaytansinoids and cell-binding agents linked via non-cleavable linkersare extremely potent, and in many cases have unexpected advantages overconjugates of maytansinoids and cell-binding agents with cleavablelinkers.

SUMMARY OF THE INVENTION

Illustrative, non-limiting embodiments of the present inventiondescribed below overcome the above disadvantages and other disadvantagesnot described above. Also, the present invention is not required toovercome the disadvantages described above, and an illustrative,non-limiting embodiment of the present invention described below may notovercome any of the problems described above.

One aspect of the present invention is a method for targeting amaytansinoid to a selected cell population comprising contacting a cellpopulation or tissue suspected of containing cells from said selectedcell population with a cell-binding agent maytansinoid conjugate,wherein one or more maytansinoids is linked to the cell-binding agentvia a non-cleavable linker.

Another aspect of the present invention is a method for treatment oftumors, autoimmune diseases, graft rejections, graft versus hostdisease, viral infections, parasite infections, and other diseases thatcan be treated by targeted therapy wherein the targeting agent is acell-binding agent, said method comprising administering to a subject inneed of treatment an effective amount of a cell-binding agentmaytansinoid conjugate wherein one or more maytansinoids is linked tothe cell-binding agent, or a pharmaceutically acceptable formulation orsolvate of said conjugate.

Another aspect of the present invention is a cell-binding agentmaytansinoid conjugate, wherein one or more maytansinoids is linked to acell-binding agent via a non-cleavable linker.

Another aspect of the present invention is a composition comprising theabove-described conjugate.

Another aspect of the present invention is a method of making theabove-described conjugate.

Another aspect of the present invention is novel maytansinoid esters.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of SMCC.

FIG. 2 shows the structure of DM1.

FIG. 3 shows graphically results of a FACS binding assay comparinghuC242 antibody to the antibody-maytansinoid conjugate huC242-SMCC-DM1.

FIG. 4 shows graphically the cytotoxicity of huC242-SMCC-DM1.

FIG. 5 shows size exclusion chromatography for huC242-SMCC-DM1.

FIGS. 6A-C and FIG. 7 show graphically the cytotoxicity ofhuC242-SMCC-DM1 compared to conjugates prepared withdisulfide-containing linkers.

FIGS. 8A-D show graphically the cytotoxicity of SMCC-DM1 conjugateslinked to various cell-binding agents.

FIG. 9 shows graphically the cytotoxicity of antibody-maytansinoidconjugate huC242-SIAB-DM1.

FIG. 10A shows graphically the antitumor activity of huC242-SMCC-DM1against COLO205 human colon cancer xenografts in SCID mice.

FIG. 10B shows graphically the antitumor activity of huC242-SMCC-DM1against SNU16 human gastric tumor xenografts in SCID mice.

FIG. 10C shows graphically the anti-tumor efficacy oftrastuzumab-SMCC-DM1 against human MCF7 tumor xenografts in SCID mice.

FIG. 11 shows graphically plasma clearance rates of huC242-SMCC-DM1compared to conjugates prepared with disulfide-containing linkers.

FIGS. 12A-D show graphically results of acute toxicity studies ofhuC242-SMCC-DM1 compared to conjugates prepared withdisulfide-containing linkers.

FIG. 13 shows the durability of cell-cycle arrest and cell destroyingactivity demonstrated by huC242-SMCC-DM1 compared to conjugates preparedwith disulfide-containing linkers.

FIGS. 14A-C show the minimal bystander effect activity ofhuC242-SMCC-DM1 compared to conjugates prepared withdisulfide-containing linkers.

FIG. 15 shows representative structures of maleimido-based cross-linkingagents.

FIG. 16 shows representative structures of haloacetyl-basedcross-linking agents.

FIG. 17 shows the structure of antibody-SMCC-DM1 conjugates.

FIG. 18 shows the structure of antibody-SIAB-DM1 conjugates.

FIG. 19 shows the structure of antibody-SMCC-DM4 conjugates.

FIG. 20 shows the structure of antibody-SIAB-DM4 conjugates.

FIG. 21 shows the synthesis of a maytansinoid cell-binding agentconjugate linked via a non-S-containing non-cleavable linker.

FIG. 22 shows graphically cytotoxicity of huC242-non-S-containingnon-cleavable linker-DM1.

FIG. 23 shows graphically results of a FACS binding assay ofhuC242-non-S-containing non-cleavable linker-DM1.

FIG. 24 shows graphically results of a HER2 ECD plate-binding assaycomparing trastuzumab antibody to the antibody-maytansinoid conjugatetrastuzumab-SMCC-DM1.

FIG. 25 shows graphically the cytotoxicity and specificity oftrastuzumab-SMCC-DM1.

FIG. 26 shows size exclusion chromatography for trastuzumab-SMCC-DM1.

FIG. 27 shows graphically results of a HER2 ECD plate-binding assaycomparing trastuzumab antibody to the antibody-maytansinoid conjugatetrastuzumab-SIAB-DM1.

FIG. 28 shows graphically the cytotoxicity and specificity oftrastuzumab-SIAB-DM1.

FIG. 29 shows size exclusion chromatography for trastuzumab-SIAB-DM1.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE INVENTION

The art reveals that it is extremely difficult to modify existing drugswithout diminishing their cytotoxic potential. However, U.S. Pat. Nos.6,441,163 B1, 6,333,410 B1, 5,416,064, and 5,208,020 demonstrate thatpotent cytotoxic agents can be created by linking maytansinoids toappropriate cell-binding agents via cleavable linkers, especiallycleavable linkers containing disulfide groups. Cell-binding agentmaytansinoid conjugates permit the full measure of the cytotoxic actionof the maytansinoids to be applied in a targeted fashion againstunwanted cells only, thereby avoiding side effects due to damage tonon-targeted, healthy cells.

The present inventors have unexpectedly discovered that maytansinoidslinked to cell-binding agents via non-cleavable linkers are superior inseveral important respects to maytansinoids linked via cleavablelinkers. In particular, when compared to conjugates containing cleavablelinkers, conjugates with non-cleavable linkers show equivalent antitumoractivity both in vitro and in vivo, but demonstrate a marked decrease inplasma clearance rate and in toxicity.

Thus, this invention provides an improved method for targeting cells,especially cells that are to be destroyed, such as tumor cells(particularly solid tumor cells), virus infected cells, microorganisminfected cells, parasite infected cells, autoimmune cells (cells thatproduce autoantibodies), activated cells (those involved in graftrejection or graft vs. host disease), or any other type of diseased orabnormal cells, while exhibiting a minimum of side effects.

The conjugate used in the inventive method has one or more maytansinoidslinked to a cell-binding agent via a non-cleavable linker. In one methodof making the conjugate, a cell-binding agent, for example an antibody,is first modified with a cross-linking reagent such as SMCC. In a secondstep, a reactive maytansinoid having a thiol group, such as DM1, isreacted with the modified antibody to produce antibody-maytansinoidconjugates. Alternatively, the maytansinoid can be modified with across-linking reagent before being reacted with a cell-binding agent.See, for example, U.S. Pat. No. 6,441,163 B1.

Suitable Maytansinoids

Maytansinoids suitable for use in the present invention are well knownin the art, and can be isolated from natural sources according to knownmethods, produced using genetic engineering techniques (see Yu et al.,99 PNAS 7968-7973 (2002)), or prepared synthetically according to knownmethods.

Examples of suitable maytansinoids include maytansinol and maytansinolanalogues. Examples of suitable maytansinol analogues include thosehaving a modified aromatic ring and those having modifications at otherpositions.

Specific examples of suitable maytansinol analogues having a modifiedaromatic ring include:

(1) C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared by LAH reductionof ansamytocin P2);

(2) C-20-hydroxy (or C-20-demethyl) +/−C-19-dechloro (U.S. Pat. Nos.4,361,650 and 4,307,016) (prepared by demethylation using Streptomycesor Actinomyces or dechlorination using LAH); and

(3) C-20-demethoxy, C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No.4,294,757) (prepared by acylation using acyl chlorides).

Specific examples of suitable maytansinol analogues having modificationsof other positions include:

(1) C-9-SH (U.S. Pat. No. 4,424,219) (prepared by the reaction ofmaytansinol with H₂S or P₂S₅);

(2) C-14-alkoxymethyl (demethoxy/CH₂OR) (U.S. Pat. No. 4,331,598);

(3) C-14-hydroxymethyl or acyloxymethyl (CH₂OH or CH₂OAc) (U.S. Pat. No.4,450,254) (prepared from Nocardia);

(4) C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (prepared by theconversion of maytansinol by Streptomyces);

(5) C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated fromTrewia nudlflora);

(6) C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (preparedby the demethylation of maytansinol by Streptomyces); and

(7) 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by the titaniumtrichloride/LAH reduction of maytansinol).

Many positions on maytansinol are known to be useful as the linkageposition, depending upon the type of link. For example, for forming anester linkage, the C-3 position having a hydroxyl group, the C-14position modified with hydroxymethyl, the C-15 position modified with ahydroxyl group and the C-20 position having a hydroxyl group are allsuitable. However the C-3 position is preferred and the C-3 position ofmaytansinol is especially preferred.

According to the present invention, a preferred maytansinoid has a freethiol group. Particularly preferred maytansinoids comprising a freethiol group include N-methyl-alanine-containing esters andN-methyl-cysteine-containing esters of maytansinol are C-3 esters ofmaytansinol and its analogs. Preferred esters includeN-methyl-alanine-containing esters and N-methyl-cysteine-containingesters of maytansinol. Synthesis of esters of maytansinol having a freethiol group has been previously described, for example in U.S. Pat. No.5,208,020, Chari et al., 52 Cancer Res., 127-131 (1992), and Liu et al.,93 Proc Natl. Acad. Sci., 8618-8623 (1996). Furthermore, U.S. Pat. No.6,333,410 B1, the entire disclosure of which is hereby incorporated byreference, provides an improved process for the preparation andpurification of thiol-containing maytansinoids suitable for linking tocell-binding agents.

Many of the conjugates of the present invention exemplified belowutilize the thiol-containing maytansinoid DM1, formally termedN^(2′)-deacetyl-N^(2′)-(3-mercapto-1-oxopropyl)-maytansine. DM1 isrepresented by the following structural formula:

The synthesis of thiol-containing maytansinoid DM1 has been previouslydescribed (U.S. Pat. No. 5,208,020).

U.S. patent application Ser. No. 10/849,136, the entire disclosure ofwhich is hereby incorporated by reference, describes sterically hinderedthiol-containing maytansinoids that bear one or two alkyl substituentson the α-carbon bearing the thiol functionality. In addition, the acylgroup of the acylated amino acid side chain of the maytansinoid bearingthe sulfhydryl group possesses a linear chain length of at least threecarbon atoms between the carbonyl group of the amide and the sulfuratom. These novel maytansinoids are suitable for use in the presentinvention.

The synthesis of maytansinoids having a sterically hindered thiol groupcan be described by reference to U.S. patent application Ser. No.10/849,136, especially FIG. 3 therein.

In one aspect of the invention, the maytansinoid contains a stericallyhindered thiol group and is represented by formula (II′-L), (II′-D), or(II′-D,L):

In the formula (II′),

-   Y₁′ represents-   (CR₇R₈)_(l)(CR₉═CR₁₀)_(p)(C≡C)_(q)A_(o)(CR₅R₆)_(m)D_(u)(CR₁₁═CR₁₂)_(r)(C≡C)_(s)B_(t)(CR₃R₄)_(n)CR₁R₂SH.

A, B, and D, each independently is cyclic alkyl or cyclic alkenyl having3 to 10 carbon atoms, simple or substituted aryl, or heterocyclicaromatic or heterocycloalkyl radical.

R₁ to R₁₂ are each independently linear alkyl or alkenyl having 1 to 10carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbonatoms, phenyl, substituted phenyl or heterocyclic aromatic orheterocycloalkyl radical, and in addition, R₂ to R₁₂ can be H.

l, m, n, o, p, q, r, s, t, and u are each independently 0 or an integerof from 1 to 5, provided that at least two of l, m, n, o, p, q, r, s, tand u are not both zero.

May represents a maytansinoid that bears a side chain at C-3 hydroxyl,C-14 hydroxymethyl, C-15 hydroxyl or C-20 desmethyl.

Another maytansinoid useful in the invention is represented by formula(II-L), (II-D), or (II-D,L):

In the formula (II),

-   Y₁ represents (CR₇R₈)_(l)(CR₅R₆)_(m)(CR₃R₄)_(n)CR₁R₂SH.

R₁ to R₈ are each independently linear alkyl or alkenyl having 1 to 10carbon atoms, branched or cyclic alkyl or alkenyl having 3 to 10 carbonatoms, phenyl, substituted phenyl, heterocyclic aromatic orheterocycloalkyl radical, and in addition R₂ to R₈ can be H.

l, m and n are each independently an integer of from 1 to 5, and inaddition n can be 0.

May represents a maytansinol that bears a side chain at C-3 hydroxyl,C-14 hydroxymethyl, C-15 hydroxyl or C-20 desmethyl.

Another useful maytansinoid is represented by formula 4₁′:

wherein the substituents are as defined for formula (II′) above.

Another useful maytansinoid is represented by formula 4₁:

wherein the substituents are as defined for formula (II) above.

Preferred are any of the above-described compounds wherein R₁ is H andR₂ is methyl or R₁ and R₂ are methyl.

Especially preferred are any of the above-described compounds, whereinR₁ is H, R₂ is methyl, R₅, R₆, R₇ and R₈ are each H, l and m are each 1,and n is 0; and those wherein R₁ and R₂ are methyl, R₅, R₆, R₇, R₈ areeach H, l and m are 1, and n is 0.

Further, the L-aminoacyl stereoisomer is preferred.

Examples of linear alkyls or alkenyls having 1 to 10 carbon atomsinclude, but are not limited to, methyl, ethyl, propyl, butyl, pentyl,hexyl, propenyl, butenyl and hexenyl.

Examples of branched alkyls or alkenyls having 3 to 10 carbon atomsinclude, but are not limited to, isopropyl, isobutyl, sec.-butyl,tert-butyl, isopentyl, 1-ethyl-propyl, isobutenyl and isopentenyl.

Examples of cyclic alkyls or alkenyls having from 3 to 10 carbon atomsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cyclopentenyl, and cyclohexenyl.

Simple aryls include aryls having 6 to 10 carbon atoms, and substitutedaryls include aryls having 6 to 10 carbon atoms bearing at least onealkyl substituent containing from 1 to 4 carbon atoms, or alkoxysubstituent such as methoxy, ethoxy, or a halogen substituent or a nitrosubstituent.

Examples of simple aryl that contain 6 to 10 carbon atoms include, butare not limited to, phenyl and naphthyl.

Examples of substituted aryl include, but are not limited to,nitrophenyl, dinitrophenyl.

Heterocyclic aromatic radicals include groups that have a 3 to10-membered ring containing one or two heteroatoms selected from N, O orS.

Examples of heterocyclic aromatic radicals include, but are not limitedto, pyridyl, nitro-pyridyl, pyrollyl, oxazolyl, thienyl, thiazolyl, andfuryl.

Heterocycloalkyl radicals include cyclic compounds, comprising 3 to10-membered ring systems, containing one or two heteroatoms, selectedform N, O or S.

Examples of heterocycloalkyl radicals include, but are not limited to,dihydrofuryl, tetrahydrofuryl, pyrrolidinyl, piperidinyl, piperazinyl,and morpholine.

Particularly preferred maytansinoids comprising a side chain thatcontains a sterically hindered thiol bond are maytansinoidsN^(2′)-deacetyl-N-^(2′)(4-mercapto-1-oxopentyl)-maytansine (termed DM3)and N^(2′)-deacetyl-N^(2′)-(4-methyl-4-mercapto-1-oxopentyl)-maytansine(termed DM4). DM3 and DM4 are represented by the following structuralformulae:

Cell-Binding Agents

The effectiveness of the compounds of the invention as therapeuticagents depends on the careful selection of an appropriate cell-bindingagent. Cell-binding agents may be of any kind presently known, or thatbecome known and include peptides and non-peptides. Generally, these canbe antibodies (especially monoclonal antibodies), lymphokines, hormones,growth factors, vitamins, nutrient-transport molecules (such astransferrin), or any other cell-binding molecule or substance thatspecifically binds a target.

More specific examples of cell-binding agents that can be used include:

polyclonal and monoclonal antibodies, including fully human antibodies;

single chain antibodies (polyclonal and monoclonal);

fragments of antibodies (polyclonal and monoclonal) such as Fab, Fab′,F(ab′)₂, and Fv (Parham, 131 J. Immunol. 2895-2902 (1983); Spring etal., 113 J. Immunol. 470-478 (1974); Nisonoff et al., 89 Arch. Biochem.Biophys. 230-244 (1960));

chimeric antibodies and antigen-binding fragments thereof;

domain antibodies (dAbs) and antigen-binding fragments thereof,including camelid antibodies (Desmyter et al., 3 Nature Struct. Biol,752, 1996);

shark antibodies called new antigen receptors (IgNAR) (Greenberg et al.,374 Nature, 168, 1995; Stanfield et al. 305 Science 1770-1773, 2004);

interferons (e.g. alpha, beta, gamma);

lymphokines such as IL-2, IL-3, IL-4, IL-6;

hormones such as insulin, TRH (thyrotropin releasing hormone), MSH(melanocyte-stimulating hormone), steroid hormones, such as androgensand estrogens;

growth factors and colony-stimulating factors such as EGF, TGF-alpha,FGF, VEGF, G-CSF, M-CSF and GM-CSF (Burgess, 5 Immunology Today 155-158(1984));

transferrin (O'Keefe et al., 260 J. Biol. Chem. 932-937 (1985)); and

vitamins, such as folate.

Monoclonal antibody techniques allow for the production of extremelyspecific cell-binding agents in the form of specific monoclonalantibodies. Particularly well known in the art are techniques forcreating monoclonal antibodies produced by immunizing mice, rats,hamsters or any other mammal with the antigen of interest such as theintact target cell, antigens isolated from the target cell, whole virus,attenuated whole virus, and viral proteins such as viral coat proteins.Sensitized human cells can also be used. Another method of creatingmonoclonal antibodies is the use of phage libraries of scFv (singlechain variable region), specifically human scFv (see e.g., Griffiths etal., U.S. Pat. Nos. 5,885,793 and 5,969,108; McCafferty et al., WO92/01047; Liming et al., WO 99/06587). In addition, resurfacedantibodies disclosed in U.S. Pat. No. 5,639,641 may also be used, as mayhumanized antibodies.

Selection of the appropriate cell-binding agent is a matter of choicethat depends upon the particular cell population that is to be targeted,but in general human monoclonal antibodies are preferred if anappropriate one is available.

For example, the monoclonal antibody J5 is a murine IgG2a antibody thatis specific for Common Acute Lymphoblastic Leukemia Antigen (CALLA)(Ritz et al, 283 Nature 583-585 (1980)) and can be used if the targetcells express CALLA such as in the disease of acute lymphoblasticleukemia.

The monoclonal antibody MY9 is a murine IgG₁ antibody that bindsspecifically to the CD33 antigen (J. D. Griffin et al 8 Leukemia Res.,521 (1984)) and can be used if the target cells express CD33 as in thedisease of acute myelogenous leukemia (AML).

Similarly, the monoclonal antibody anti-B4 interchangeably also calledB4, is a murine IgG₁ that binds to the CD19 antigen on B cells (Nadleret al, 131 J. Immunol. 244-250 (1983)) and can be used if the targetcells are B cells or diseased cells that express this antigen such as innon-Hodgkin's lymphoma or chronic lymphoblastic leukemia.

In addition, the monoclonal antibody C242 that binds to the CanAgantigen (U.S. Pat. No. 5,552,293) can be used to treat CanAg expressingtumors, such as colorectal, pancreatic, non-small cell lung, and gastriccancers. HuC242 is a humanized form of the monoclonal antibody C242 thatis described in U.S. Pat. No. 5,552,293 and for which the hybridoma isdeposited with the ECACC identification Number 90012601. A humanizedform can be prepared by either applying the CDR-grafting methodology(U.S. Pat. Nos. 5,585,089; 5,693,761; and 5,693,762) or the resurfacingmethodology (U.S. Pat. No. 5,639,641). HuC242 can also be used to treatCanAg expressing tumors, such as colorectal, pancreatic, non-small celllung, and gastric cancers.

Further, the antibody trastuzumab can be used to treat breast and othercancers, such as prostate and ovarian cancers that express the Her2antigen.

Anti-IGF-IR antibodies that bind to insulin growth factor receptor arealso useful.

Ovarian cancer and prostate cancer can be successfully targeted with,for example, an anti-MUC1 antibody, such as anti-HMFG-2(Taylor-Papadimitriou et al., 28. Int. J. Cancer 17-21, 1981) or hCTM01(56 Cancer Res. 5179-5185, 1996) and an anti-PSMA (prostate-specificmembrane antigen), such as J591 (Liu et al. 57 Cancer Res. 3629-3634,1997) respectively.

Non-antibody molecules can also be used to target specific cellpopulations. For example, GM-CSF, which binds to myeloid cells, can beused as a cell-binding agent to target diseased cells from acutemyelogenous leukemia. In addition, IL-2, which binds to activatedT-cells, can be used for prevention of transplant graft rejection, fortherapy and prevention of graft-versus-host disease, and for treatmentof acute T-cell leukemia. MSH, which binds to melanocytes, can be usedfor the treatment of melanoma. Folic acid can be used to target thefolate receptor expressed on ovarian and other tumors. Epidermal growthfactor (EGF) can be used to target squamous cancers such as lung andhead and neck. Somatostatin can be used to target neuroblastomas andother tumor types. Cancers of the breast and testes can be successfullytargeted with estrogen (or estrogen analogues) or androgen (or androgenanalogues) respectively as cell-binding agents.

Cross-Linking Reagents

The maytansinoid is linked to the cell-binding agent by means of across-linking reagent that, when reacted, forms a non-cleavable linkerbetween the maytansinoid and the cell-binding agent.

As used herein, a “linker” is any chemical moiety that links acell-binding agent covalently to a maytansinoid. In some instances, partof the linker is provided by the maytansinoid. For example, DM1, athiol-containing maytansinoid (FIG. 2), is a derivative of the naturalmaytansinoid, maytansine, and provides part of the linker. The sidechain at the C-3 hydroxyl group of maytansine ends in —CO—CH₃, the sidechain of DM1 ends in —CO—CH₂—CH₂—SH. Therefore the final linker isassembled from two pieces, the cross-linking reagent introduced into thecell-binding agent and the side chain from the DM1.

Cleavable linkers are linkers that can be cleaved under mild conditions,i.e. conditions under which the activity of the maytansinoid drug is notaffected. Many known linkers fall in this category and are describedbelow.

Disulfide containing linkers are linkers cleavable through disulfideexchange, which can occur under physiological conditions.

Acid-labile linkers are linkers cleavable at acid pH. For example,certain intracellular compartments, such as endosomes and lysosomes,have an acidic pH (pH 4-5), and provide conditions suitable to cleaveacid-labile linkers.

Linkers that are photo-labile are useful at the body surface and in manybody cavities that are accessible to light. Furthermore, infrared lightcan penetrate tissue.

Some linkers can be cleaved by peptidases. Only certain peptides arereadily cleaved inside or outside cells, see e.g. Trouet et al., 79Proc. Natl. Acad. Sci. USA, 626-629 (1982) and Umemoto et al. 43 Int. J.Cancer, 677-684 (1989). Furthermore, peptides are composed of α-aminoacids and peptidic bonds, which chemically are amide bonds between thecarboxylate of one amino acid and the α-amino group of a second aminoacid. Other amide bonds, such as the bond between a carboxylate and theε-amino group of lysine, are understood not to be peptidic bonds and areconsidered non-cleavable.

Some linkers can be cleaved by esterases. Again only certain esters canbe cleaved by esterases present inside or outside cells. Esters areformed by the condensation of a carboxylic acid and an alcohol. Simpleesters are esters produced with simple alcohols, such as aliphaticalcohols, and small cyclic and small aromatic alcohols. For example, thepresent inventors found no esterase that cleaved the ester at C-3 ofmaytansine, since the alcohol component of the ester, maytansinol, isvery large and complex.

A non-cleavable linker is any chemical moiety that is capable of linkinga maytansinoid to a cell-binding agent in a stable, covalent manner anddoes not fall under the categories listed above as cleavable linkers.Thus, non-cleavable linkers are substantially resistant to acid-inducedcleavage, light-induced cleavage, peptidase-induced cleavage,esterase-induced cleavage, and disulfide bond cleavage.

“Substantially resistant” to cleavage means that the chemical bond inthe linker or adjoining the linker in at least 80%, preferably at least85%, more preferably at least 90%, even more preferably at least 95%,and most preferably at least 99% of the cell-binding agent maytansinoidconjugate population remains non-cleavable by an acid, aphotolabile-cleaving agent, a peptidase, an esterase, or a chemical or aphysiological compound that cleaves the chemical bond (such as adisulfide bond) in a cleavable linker, for within a few hours to severaldays of treatment with any of the agents described above.

Furthermore, “non-cleavable” refers to the ability of the chemical bondin the linker or adjoining to the linker to withstand cleavage inducedby an acid, a photolabile-cleaving agent, a peptidase, an esterase, or achemical or a physiological compound that cleaves a disulfide bond, atconditions under which the maytansinoid or the cell binding agent doesnot lose its activity.

A person of ordinary skill in the art would readily distinguishnon-cleavable from cleavable linkers.

An example of an appropriate control for testing whether a linker issubstantially resistant to cleavage is a linker with a chemical bond,such as a disulfide bond, that is susceptible to cleavage by any of theagents described above. One can test whether a linker is substantiallyresistant to cleavage by measuring the stability of the conjugates byELISA, HPLC, or other suitable means, over a period of time extendingfrom between a few hours to several days, typically 4 hours to 5 days.ELISA assays can be used to measure the level of stable conjugate in theplasma concentration.

Non-cleavable linkers are also characterized in that the in vivohalf-life of conjugates comprising non-cleavable linkers is generallyabout 20% higher than that of conjugates comprising cleavable linkers.In mice, the in vivo half-life of IgG-maytansinoid conjugates linked vianon-cleavable linkers is at least 4 days.

Suitable cross-linking reagents that form non-cleavable linkers betweenthe maytansinoid and the cell-binding agent are well known in the art,and can form non-cleavable linkers that comprise a sulfur atom (such asSMCC) or that are without a sulfur atom.

Preferred cross-linking reagents that form non-cleavable linkers betweenthe maytansinoid and the cell-binding agent comprise a maleimido- orhaloacetyl-based moiety. According to the present invention, suchnon-cleavable linkers are said to be derived from maleimido- orhaloacetyl-based moiety. Cross-linking reagents comprising amaleimido-based moiety include N-succinimidyl4-(maleimidomethyl)cyclohexanecarboxylate (SMCC),N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate),which is a “long chain” analog of SMCC (LC-SMCC), κ-maleimidoundecanoicacid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N-succinimidylester (GMBS), ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),N-(α-maleimidoacetoxy)-succinimide ester [AMAS],succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl4-(p-maleimidophenyl)-butyrate (SMPB), andN-(p-maleimidophenyl)isocyanate (PMPI) (see FIG. 15 for representativestructures of maleimido-based cross-linking reagents). Thesecross-linking reagents form non-cleavable linkers derived frommaleimido-based moieties.

Cross-linking reagents comprising a haloacetyl-based moiety includeN-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyliodoacetate (SIA), N-succinimidyl bromoacetate (SBA) and N-succinimidyl3-(bromoacetamido)propionate (SBAP) (see FIG. 16 for representativestructures of haloacetyl-based cross-linking agents). Thesecross-linking reagents form non-cleavable linkers derived fromhaloacetyl-based moieties.

While the active esters described in FIGS. 15 and 16 are comprised ofN-succinimidyl and sulfosuccinimidyl esters, other active esters, suchas N-hydroxy phthalimidyl esters, N-hydroxy sulfophthalimidyl esters,ortho-nitrophenyl esters, para-nitrophenyl esters, 2,4-dinitrophenylesters, 3-sulfonyl-4-nitrophenyl esters, 3-carboxy-4-nitrophenyl esters,pentaflurophenyl esters, and sulfonyl tetrafluorophenyl esters can alsobe used.

Particularly preferred cross-linking reagents form non-cleavable linkersthat do not contain a sulfur atom. FIG. 21 shows a maytansinoid moleculederivatized with a cross-linking reagent that is derived from anα,ω-dicarboxylic acid (an alkane or alkene dioic acid wherein the alkaneor alkene has 3-24 carbon atoms). When reacted with the cell-bindingagent, the cross-linking reagent will form a non-sulfur containingnon-cleavable linker (non-S-containing non-cleavable linker).

The maytansinoid molecule of FIG. 21 is made as follows. First amonoester of adipic acid (also known as hexanedioic acid or1,6-hexanedicarboxylic acid) is prepared by treatment with oneequivalent of 2-trimethysilylethanol in the presence ofdicyclohexylcarbodiimide. Activation of the remaining carboxylic acidgroup with isobutyl chloroformate, followed by reaction withN-methyl-L-alanine, provides the acylated N-methyl-L-alanine. Reactionwith maytansinol in the presence of dicyclohexylcarbodiimide and zincchloride, followed by removal of the trimethylsilyl protecting groupwith tetrabutylammonium fluoride, provides the maytansinoid esterbearing a free carboxy group. Esterification of the carboxyl group byreaction with sulfo N-hydroxysuccinimide in the presence ofdicyclohexylcarbodiimide provides the active ester of maytansinol thatcan react with a cell-binding agent to give a non-cleavable conjugatethat does not contain a sulfur atom.

Non-cleavable linkers that do not contain a sulfur atom can also bederived from other dicarboxylic acid based moieties using the methoddescribed above. Other suitable dicarboxylic acid based moieties includebut are not limited to α,ω-dicarboxylic acids of general formula (IV):HOOC—X_(l)—Y_(n)—Z_(m)—COOH  (IV)

In formula (IV), X is a linear or branched alkyl, alkenyl or alkynylgroup having 2 to 20 carbon atoms, Y is a cycloalkyl or cycloalkenylgroup bearing 3 to 10 carbon atoms, Z is a substituted or unsubstitutedaromatic group bearing 6 to 10 carbon atoms or a substituted orunsubstituted heterocyclic group wherein the hetero atom is selectedfrom N, O or S, and wherein l, m and n are each 0 or 1, provided thatthey are all not 0 at the same time.

Maytansinoids derivatized to contain an active ester that can bedirectly reacted with a cell-binding agent to form a conjugate having anon-S-containing non-cleavable linker can be represented by formula 5:

wherein X, Y, Z, l, m and n are all defined as for formula (IV) above,and further wherein E together with the carbonyl group forms an activeester such as N-hydroxy succinimidyl and sulfosuccinimidyl esters,N-hydroxy phthalimidyl ester, N-hydroxy sulfophthalimidyl ester,ortho-nitrophenyl ester, para-nitrophenyl ester, 2,4-dinitrophenylester, 3-sulfonyl-4-nitrophenyl ester, 3-carboxy-4-nitrophenyl ester,pentaflurophenyl ester, and sulfonyl tetrafluorophenyl ester.

Preferred is a derivatized maytansinoid represented by formula 6:

wherein n represents an integer from 3 to 24, and E has the samedefinition as for the maytansinoid of formula 5.

A more preferred embodiment is the derivatized maytansinoid representedby formula 7:

wherein R is H or SO₃ ⁻Na⁺.

Compounds of the formulae 5, 6, and 7 are novel maytansinoids.

Examples of linear alkyl, alkenyl, or alkynyl groups having 2 to 20carbon atoms include, but are not limited to, ethyl, propyl, butyl,pentyl, hexyl, propenyl, butenyl, and hexenyl.

Examples of branched alkyl, alkenyl, or alkynyl groups having 2 to 20carbon atoms include, but are not limited to, isopropyl, isobutyl,sec.-butyl, tert.-butyl, isopentyl, 1-ethyl-propyl, isobutenyl,isopentenyl, ethynyl, propynyl (propargyl), 1-butynyl, 2-butynyl, and1-hexynyl.

Examples of cycloalkyl or cycloalkenyl groups having from 3 to 10 carbonatoms include, but are not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclopentenyl, cyclohexenyl, andcycloheptadienyl.

Examples of aromatic groups that contain 6 to 10 carbon atoms include,but are not limited to, phenyl and naphthyl.

Examples of substituted aromatic groups include, but are not limited to,nitrophenyl and dinitrophenyl.

Heterocyclic aromatic groups include, but are not limited to, groupsthat have a 3 to 10-membered ring containing one or two heteroatomsselected from N, O or S.

Examples of substituted and unsubstituted heterocyclic aromatic groupsinclude, but are not limited to, pyridyl, nitro-pyridyl, pyrollyl,oxazolyl, thienyl, thiazolyl, and furyl.

Heterocycloalkyl radicals include, but are not limited to, cycliccompounds, comprising 3 to 10-membered ring systems, containing one ortwo heteroatoms, selected from N, O or S.

Examples of heterocycloalkyl radicals include, but are not limited to,dihydrofuryl, tetrahydrofuryl, tetrahydropyrollyl, piperidinyl,piperazinyl, and morpholino.

Examples of α,ω-dicarboxylic acids of the general formulaHOOC—X_(l)—Y_(n)—Z_(m)—COOH include, but are not limited to, adipicacid, glutaric acid, pimelic acid, hexene-1,6-dioc acid,pentene-1,5-dioc acid, cyclohexane-dioic acid, and cyclohexene-dioicacid

Synthesis of Cytotoxic Conjugates

Conjugates of cell-binding agents and maytansinoids can be formed usingany techniques presently known or later developed.

Methods of conjugation of cell-binding agents with maytansinoidsgenerally involve two reaction steps. In one method, described in U.S.Pat. No. 5,208,020, a cell-binding agent, such as an antibody, can bemodified with a cross-linking reagent to introduce one or more, usually1-10, reactive groups. The modified cell-binding agent is then reactedwith one or more thiol-containing maytansinoids to produce a conjugate.

Alternatively, as disclosed in U.S. Pat. No. 6,441,163 B1, athiol-containing maytansinoid can first be modified with a cross-linkingreagent, followed by reaction of the modified maytansinoid with acell-binding agent. For example, the thiol-containing maytansinoid canbe reacted with the maleimido compounds described in FIG. 15 or with thehaloacetyl compounds described in FIG. 16, to give a maytansinoidthioether bearing an active succinimidyl or sulfosuccinimidyl ester.Reaction of these maytansinoids containing an activated linker moietywith a cell-binding agent provides another method of producing anon-cleavable cell-binding agent maytansinoid conjugate.

In another aspect of the invention, as disclosed above, a maytansinoidthat does not contain a sulfur atom can first be derivatized by adicarboxylic acid based cross-linking reagent, followed by reaction withthe cell-binding agent, to form a conjugate in which the maytansinoid islinked to the cell-binding agent via a non-S-containing non-cleavablelinker.

Typically, an average of 1-10 maytansinoids per antibody are linked. Theconjugate can be purified through a Sephadex G-25 column.

The entire disclosures of U.S. Pat. Nos. 5,208,020 and 6,441,163 B1 areexpressly incorporated herein by reference.

Representational conjugates of the invention are antibody-maytansinoidderivatives, antibody fragment-maytansinoid derivatives, growthfactor-maytansinoid conjugates, such as epidermal growth factor(EGF)-maytansinoid derivatives, hormone-maytansinoid conjugates, such asmelanocyte stimulating hormone (MSH)-maytansinoid derivatives, thyroidstimulating hormone (TSH)-maytansinoid derivatives,estrogen-maytansinoid derivatives, estrogen analogue-maytansinoidderivatives, androgen-maytansinoid derivatives, androgenanalogue-maytansinoid derivatives, and vitamin-maytansinoid conjugates,such as folate maytansinoid.

Maytansinoid conjugates of antibodies, antibody fragments, proteinhormones, protein growth factors and other proteins are made in the sameway. For example, peptides and antibodies can be modified with thenon-cleavable cross-linking reagents mentioned above. A solution of anantibody in aqueous buffer may be incubated with a molar excess of anantibody-modifying cross-linking reagent such as succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), sulfo-SMCC,-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfo-MBS,succinimidyl-iodoacetate, or N-succinimidyl-4-(iodoacetyl)-aminobenzoate(SIABN-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate),which is a “long chain” analog of SMCC (LC-SMCC), sulfo-LC-SMCC,κ-maleimidoundecanoic acid N-succinimidyl ester (KMUA), sulfo-KMUA,γ-maleimidobutyric acid N-succinimidyl ester (GMBS), sulfo-GMBS,ε-maleimidcaproic acid N-hydroxysuccinimide ester (EMCS), sulfo-EMCS,N-(α-maleimidoacetoxy)-succinimide ester (AMAS), sulfo-AMAS,succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH), sulfo-SMPH,N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), sulfo-SMPH,N-(p-maleimidophenyl)isocyanate (PMPI),N-succinimidyl-4-(iodoacetyl)-aminobenzoate (STAB), N-succinimidyliodoacetate (SIA), N-succinimidyl bromoacetate (SBA) and N-succinimidyl3-(bromoacetamido)propionate (SBAP), as described in the literature.See, Yoshitake et al., 101 Eur. J. Biochem. 395-399 (1979); Hashida etal., J. Applied Biochem. 56-63 (1984); and Liu et al., 18 690-697(1979); Uto et al., 138 J. Immunol. Meth. 87-94 (1991); Rich et al. 18J. Med. Chem. 1004-1010 (1975); Kitagawa and Aikawa, 79 J. Biochem.(Tohyo) 233-236 (1976); Tanimori et al., 62 J. Immunol. Meth. 123-128(1983); Hashida et al., 6 J. Appl. Biochem. 56-63 (1984); Thorpe et al.,140 Eur. J. Biochem. 63-71 (1984), Chrisey et al. 24 Nucl. Acid Res.3031-3039 (1996), Annunziato et al., 4 Bioconjugate Chem. 212-218(1993), Rector et al., 24 J. Immunol. Meth. 321-336 (1978), and Inman etal. 2 Bioconjugate. Chem. 458-463 (1991).

The modified antibody is then treated with the thiol-containingmaytansinoid (1.25 molar equivalent/maleimido or iodoacetyl group) toproduce a conjugate. The mixtures are incubated overnight at about 4° C.The antibody-maytansinoid conjugates are purified by gel filtrationthrough a Sephadex G-25 column. The number of maytansinoid moleculesbound per antibody molecule can be determined by measuringspectrophotometrically the ratio of the absorbance at 252 nm and 280 nm.Typically, an average of 1-10 maytansinoids per antibody are linked.

A preferred method is to modify antibodies with succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) to introducemaleimido groups followed by reaction of the modified antibody with athiol-containing maytansinoid to give a thioether-linked conjugate.Again, conjugates with 1 to 10 drug molecules per antibody moleculeresult. Examples of antibody-maytansinoid conjugates are shown in FIGS.17-20.

Similarly, for example, estrogen and androgen cell-binding agents suchas estradiol and androstenediol can be esterified at the C-17 hydroxygroup by reaction with an appropriately protected thiol group-containingcarboxylic acid chloride such as 3-S-acetylpropanoyl chloride. Othermethods of esterification can also be employed as described in theliterature (Haslam, 36 Tetrahedron 2400-2433 (1980)). The protected orfree thiol-containing androgen or estrogen can then be reacted with athiol-containing maytansinoid to produce conjugates. The conjugates canbe purified by column chromatography on silica gel or by HPLC.

A particularly preferred method is to modify maytansinol with across-linking reagent that results in a linkage that does not containany sulfur atoms, followed by reaction of the modified maytansinoid withan antibody to produce conjugates.

Therapeutic Efficacy of the Cytotoxic Conjugates of the Invention

Cell-binding agent maytansinoid conjugates of the invention can beevaluated for their ability to suppress proliferation of various celllines in vitro. For example, cell lines such as the human coloncarcinoma line COLO205, the human melanoma cell line A375, the humanmyeloid leukemia cell line HL60, the human breast carcinoma line SKBR3,or the human epidermoid carcinoma cell line KB can be used for theassessment of cytotoxicity of these conjugates. Cells to be evaluatedcan be exposed to the compounds for 24 hours and the surviving fractionsof cells measured in direct assays by known methods. (See, e.g.Goldmacher et al., 135 J. Immunol. 3648-3651 (1985), and Goldmacher etal., 102 J. Cell Biol. 1312-1319 (1986).) IC₅₀ values can then becalculated from the results of the assays.

High cytotoxicity can be defined as exhibiting a toxicity having an IC₅₀(the inhibiting concentration of a toxic substance that leaves asurviving fraction of 0.5) of about 10⁻⁸ M or less when measured invitro with SKBR3 cells upon a 24 hour exposure time to the drug.

The in vitro potency and target specificity of antibody-maytansinoidconjugates of the present invention are shown in FIG. 4. Conjugates ofhuC242 with DM1 using the cross-linking reagent SMCC are highly potentin destroying antigen positive SKBR3 cells, with an IC₅₀ value of3.5×10⁻¹² M. In contrast, antigen negative A375 cells are about 800-foldless sensitive demonstrating that maytansinoid conjugates of the presentinvention are highly potent and specific.

The huC242-SMCC-DM1 conjugate was of equal or greater potency whencompared to conjugates prepared with disulfide-containing linkers inclonogenic (FIG. 6A-C) and in indirect cytotoxicity assays (FIG. 7).These results were unexpected, based on previously published datademonstrating that an anti-Her2 antibody conjugated to maytansinoids viaSMCC showed no specific activity (Chari et al., 52 Cancer Res. 127-133(1992).

Activity of conjugates prepared with SMCC non-cleavable linker is notrestricted to huC242 conjugates. Specific activity in vitro was alsoobserved with SMCC-DM1 conjugates of trastuzumab, an anti-Her2 antibody;My9-6, an anti-CD33 antibody; KS77, an anti-EGFR antibody; and N901, ananti-CD56 antibody (FIGS. 8A-D and 25).

In addition, conjugates with non-cleavable linkers that show specificactivity in vitro are not restricted to the SMCC linker. A huC242conjugate of DM1 synthesized with the non-cleavable linker STAB showedpotent and antigen-specific cytotoxicity in clonogenic assays in vitro(FIG. 9). Further, a trastuzumab conjugate of DM1 synthesized with SIABwas also cytotoxic in clonogenic assays (FIG. 28). Further, ahuC242-non-S-containing non-cleavable linker-DM1 conjugate alsodemonstrated potent and antigen-specific cytotoxicity in clonogenicassays in vitro (FIG. 22).

Antibody conjugates with DM1 using the SMCC linker show anti-tumorefficacy against human tumor xenografts in mice (FIG. 10A-C). First, asshown in FIG. 10A, marked inhibition of tumor growth was observed upontreatment of COLO 205 colon tumor xenografts with huC242-SMCC-DM1. Inthis experiment, one group of five animals bearing establishedsubcutaneous tumors was treated with huC242-SMCC-DM1 at a dose of 150μg/kg of conjugated DM1. Tumor sizes were measured periodically andgraphed vs. time after tumor inoculation. All five treated animals had acomplete remission, although three animals relapsed thereafter atdifferent time points, whereas two animals stayed tumor free untiltermination of the experiment (FIG. 10A). This anti-tumor activity isobserved at conjugate doses that have no effect on mouse body weight, ameasure of drug toxicity. Second, as shown in FIG. 10B, treatment ofmice bearing COLO205 colon carcinoma tumor xenografts with thehuC242-SMCC-DM1 conjugate resulted in complete regression of tumors,with some mice remaining free of detectable tumors for over 2 monthspost-treatment (FIG. 10A). In this experiment, three groups of fiveanimals each bearing established subcutaneous SNU tumors were treatedwith huC242-SMCC-DM1 at doses of 15 μg/kg, 30 μg/kg, and 60 μg/kg ofconjugated DM1, respectively. Tumor sizes were measured periodically andgraphed vs. time after tumor inoculation. HuC242-SMCC-DM1 showed adose-dependent antitumor effect. Again, this activity was obtained at aconjugate concentration that showed no effect on mouse body weight. Atrastuzumab-SMCC-DM1 conjugate also showed significant tumor regressionin a mouse tumor xenograft model with the MCF-7 breast carcinoma cellline (FIG. 10C).

Plasma clearance of antibody-maytansinoid conjugate synthesized with thenon-cleavable linker SMCC is very slow and comparable to the clearanceof antibody alone. This is in sharp contrast to plasma clearance ofconjugates prepared with relatively labile disulfide bonds such ashuC242-SPP-DM1. For example, the half-life for clearance of the SMCCconjugate is approximately 320 hours, while the half-life for the SPPconjugate is in the range of 40-50 hours (FIG. 11). However, theclearance of the antibody component for each type of conjugate isidentical, suggesting that the difference in measured conjugateclearance rate is due to the loss of maytansinoid from the antibodyconjugate in the case of the SPP-DM1 conjugate. The non-cleavable SMCClinkage is therefore much more resistant to maytansinoid-linker cleavageactivities present in vivo than the SPP-DM1 conjugate. Further, thedecreased clearance rate for the SMCC linked conjugates, compared toSPP-DM1 conjugates, leads to a nearly 5-fold increase in overallmaytansinoid exposure of the animal as measured by the area under thecurve (AUC). This increased exposure could have substantial impact ondrug efficacy in some cases.

Maytansinoid conjugates prepared with non-cleavable linkers such as SMCCshow an unexpected increased tolerability in mice compared withconjugates prepared with cleavable disulfide linkers. An acute toxicitytest with a single intravenous dose was carried out in female CD-1 mice.A comparison of the tolerability of a huC242-SMCC-DM1 conjugate(non-cleavable) with huC242 conjugates prepared with linkers containingcleavable disulfide bonds was conducted by monitoring the death of mice(FIGS. 12A and B) and signs of toxicity (FIGS. 12C and D) over a seriesof four escalating doses of each conjugate. The maximum tolerated dose(MTD) for the SMCC-DM1 conjugate was greater than the highest dosetested (150 mg/kg) while the MTD for the disulfide-linked conjugateSPP-DM1 was in the range of 45-90 mg/kg. At 150 mg/kg, all mice in theSMCC-DM1 treated group survived, while lethal toxicity was observed forall mice in the SPP-DM1 treated group by 96 hours post-treatment.

Maytansinoid conjugates are thought to impart their cell destroyingactivity through the inhibition of microtubule polymerization. Thisinhibition of microtubule polymerization leads to an arrest of the cellcycle principally at G2/M. The antigen-dependent arrest of cells at G2/Mby antibody-maytansinoid conjugates can be monitored by flow cytometryanalysis (FIG. 13). Treatment of COLO205 cells with huC242-SPP-DM1 orhuC242-SMCC-DM1 conjugate results in a complete G2/M arrest by 6-10hours. By 30 hours post-treatment however, some of the cells arrested bytreatment with the disulfide-linked huC242-SPP-DM1 conjugate escape fromcell cycle arrest and reinitiate cell division. Surprisingly, cellstreated with the non-cleavable conjugate do not escape from the cellcycle block at this later time point. The difference in the durabilityof the activity of these two conjugates is also reflected in percentageof dead cells at the 30 hour time point, as judged by a dye exclusionassay using trypan blue. These results demonstrate an unexpecteddurability of the molecular events induced by treatment with thenon-cleavable SMCC linker conjugates.

An additional aspect of conjugates prepared with non-cleavable linkerscompared to conjugates that have cleavable disulfide linkers is theabsence of activity toward antigen-negative cells when in closeproximity to antigen-positive cells, termed here the bystander effect.That is, the conjugates prepared with non-cleavable linkers have minimalbystander activity. Both the huC242-SPP-DM1 (cleavable) and huC242-SMCC(non-cleavable) conjugates show potent cell destroying activity towardthe antigen-positive COLO 205 cell line and have no activity toward theantigen-negative cell line, Namalwa, when cultured separately (FIG.14A-C). However, treatment of co-cultures of COLO 205 and Namalwa cellswith huC242-SPP-DM1 reveals dramatic cell destroying activity of theconjugate toward even the antigen-negative Namalwa cells. In contrast,the huC242-SMCC-DM1 conjugate does not demonstrate any such bystanderactivity under these conditions. No cell destroying activity againstNamalwa cells is observed with the huC242-SMCC-DM1 conjugate even whenco-cultured with the antigen-positive COLO 205 cells. This minimalbystander activity of the non-cleavable conjugate, as measured in thisin vitro assay, may contribute to the increased tolerability ofconjugate with non-cleavable linkers observed in acute toxicity studies.

Results from the above experiments demonstrate that the maytansinoidconjugates with non-cleavable linkers of the present invention possessvastly improved anti-tumor activity compared to previously describedcell-binding agent maytansinoid conjugates.

Methods of Use

The above-described conjugates can be used in a method for targetingmaytansinoids to a selected cell population, the method comprisingcontacting a cell population or tissue suspected of containing theselected cell population with a cell-binding agent maytansinoidconjugate, wherein one or more maytansinoids is covalently linked to thecell-binding agent via a non-cleavable linker and the cell-binding agentbinds to cells of the selected cell population.

The above-described conjugates can also be used in a method ofdestroying cells, the method comprising contacting the cells with acell-binding agent maytansinoid conjugate, wherein one or moremaytansinoids is covalently linked to the cell-binding agent via anon-cleavable linker and the cell-binding agent binds to the cells.

The above-described conjugates can also be used in a method of treatmentof afflictions including but not limited to malignant tumors, autoimmunediseases, graft rejections, graft versus host disease, viral infections,microorganism infections, and parasite infections, the method comprisingadministering to a subject in need of treatment an effective amount of acell-binding agent maytansinoid conjugate, wherein one or moremaytansinoids is covalently linked to the cell-binding agent via anon-cleavable linker and the cell-binding agent binds diseased orinfected cells of the affliction.

Examples of medical conditions that can be treated according to themethods of the present invention include but are not limited tomalignancy of any type including, for example, cancer of the lung,breast, colon, prostate, kidney, pancreas, ovary, and lymphatic organs;autoimmune diseases, such as systemic lupus, rheumatoid arthritis, andmultiple sclerosis; graft rejections, such as renal transplantrejection, liver transplant rejection, lung transplant rejection,cardiac transplant rejection, and bone marrow transplant rejection;graft versus host disease; viral infections, such as CMV infection, HIVinfection, AIDS, etc.; and parasite infections, such as giardiasis,amoebiasis, schistosomiasis, and others as determined by one of ordinaryskill in the art.

The methods can be practiced in vitro or in vivo.

The above-described conjugates can be used in a method of in vitro useto treat, for example, autologous bone marrow cells prior to theirtransplant into the same patient in order to destroy diseased ormalignant cells; bone marrow cells or other tissue prior to theirtransplantation in order to destroy T cells and other lymphoid cells andprevent graft-versus-host-disease (GVHD); cell cultures in order todestroy all cells except for desired variants that do not express thetarget antigen; or cell cultures in order to destroy variant cells thatexpress undesired antigen; the method comprising treating the cells withan effective amount of a cell-binding agent maytansinoid conjugate,wherein one or more maytansinoids is covalently linked to thecell-binding agent via a non-cleavable linker and the cell-binding agentbinds the cells that are to be destroyed.

The conditions of clinical and non-clinical in vitro use are readilydetermined by one of ordinary skill in the art.

For example, treatment can be carried out as follows. Bone marrow can beharvested from the patient or other individual and then incubated inmedium containing serum to which is added the cytotoxic agent of theinvention, concentrations range from about 10 pM to 1 nM, for about 30minutes to about 48 hours at about 37° C. The exact conditions ofconcentration and time of incubation, i.e., the dose, can be readilydetermined by one of ordinary skill in the art. After incubation thebone marrow cells can be washed with medium containing serum andreturned to the patient intravenously according to known methods. Incircumstances where the patient receives other treatment such as acourse of ablative chemotherapy or total-body irradiation between thetime of harvest of the marrow and reinfusion of the treated cells, thetreated marrow cells can be stored frozen in liquid nitrogen usingstandard medical equipment.

For clinical in vivo use, the cytotoxic agent can be supplied as asolution or a lyophilized powder that is tested for sterility and forendotoxin levels. Examples of suitable protocols of conjugateadministration are as follows. Conjugates can be given weekly for 4weeks as an intravenous bolus each week. Bolus doses can be given in 50to 500 ml of normal saline to which 5 to 10 ml of human serum albumincan be added. Dosages will be 10 mg to 2000 mg per administration,intravenously (range of 100 ng to 20 mg/kg per day). After four weeks oftreatment, the patient can continue to receive treatment on a weeklybasis.

Specific in vivo clinical protocols with regard to route ofadministration, excipients, diluents, dosages, times, etc., can bedetermined by one of ordinary skill in the art as the clinical situationwarrants.

If desired, other active agents, such as other anti-tumor agents, may beadministered along with the conjugate.

Novel Conjugates, Compositions and Methods of Making the Conjugates

While some conjugates of antibodies and maytansinoids linked by anon-cleavable linker are known, others are new. Therefore there isprovided a cell-binding agent maytansinoid conjugate having at least onemaytansinoid linked to a cell-binding agent via a non-cleavable linker,provided that the linker does not comprise a group derived from across-linking agent selected from the group consisting of: succinimidyl4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), sulfo-SMCC,m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfo-MBS, andsuccinimidyl-iodoacetate when the cell-binding agent is an antibody.

The new conjugates can be made and used as described above.

The composition comprises the cell-binding agent maytansinoid conjugateand a carrier.

The carrier may be a pharmaceutically acceptable carrier, diluent orexcipient.

Suitable pharmaceutically acceptable carriers, diluents, and excipientsare well known and can be determined by those of ordinary skill in theart as the clinical situation warrants.

Examples of suitable carriers, diluents and/or excipients include: (1)Dulbecco's phosphate buffered saline, pH about 7.4, containing or notcontaining about 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9%saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose; and may also containan antioxidant such as tryptamine and a stabilizing agent such as Tween20.

For these new conjugates, syntheses methods are also provided.

One of the processes of making the cell-binding agent maytansinoidconjugate comprises:

(a) providing the cell-binding agent

(b) modifying the cell-binding agent with a cross-linking agent, and

(c) conjugating the modified cell-binding agent with a maytansinoid or athiol-containing maytansinoid thereby providing the non-cleavable linkerbetween the cell-binding agent and the maytansinoid or thiol-containingmaytansinoid to produce the conjugate.

Another process of making the cell-binding agent maytansinoid conjugatecomprises:

(a) providing the maytansinoid or a thiol-containing maytansinoid,

(b) modifying the maytansinoid or thiol-containing maytansinoid with across-linking agent to thereby form a non-cleavable linker, and

(c) conjugating the modified maytansinoid or thiol-containingmaytansinoid with the cell-binding agent, thereby providing thenon-cleavable linker between the cell-binding agent and the maytansinoidor thiol-containing maytansinoid to produce the conjugate.

An additional process of making the cell-binding agent maytansinoidconjugate comprises:

(a) providing the maytansinoid,

(b) modifying the maytansinoid to provide a non-sulfur-containingmaytansinol having an active ester, and

(c) conjugating the modified maytansinoid with the cell-binding agent,thereby providing a non-S-containing non-cleavable linker between thecell-binding agent and the maytansinol to produce the conjugate. Thesemethods are described in detail above and in the United States patentscited herein and expressly incorporated herein by reference.

EXAMPLES

The invention will now be illustrated by reference to non-limitingexamples. Unless otherwise stated, all percents, ratios, parts, etc. areby weight.

The buffers used in the following experiments were: 50 mM potassiumphosphate (KPi)/50 mM sodium chloride (NaCl)/2 mMethylenediaminetetraacetic acid (EDTA), pH 6.5 (Buffer A); 1× phosphatebuffered saline (PBS), pH 6.5 (Buffer B); and 0.1 M KPi buffer/2 mM EDTAat pH 7.5 (Assay Buffer).

SMCC (Product No. 22360, M.W. 334.33 g/mole) and SIAB (Product No.22329, M.W. 402.15 g/mole) were purchased from Pierce. The huC242antibody is a humanized form of the monoclonal antibody C242, describedin U.S. Pat. No. 5,552,293, for which the hybridoma is deposited withthe ECACC Identification Number 90012601). Trastuzumab antibody wasobtained from Genentech. DM1 (free thiol form; M.W. 737.5 g/mole) wasprepared as described previously in U.S. Pat. Nos. 5,208,020 and6,333,410 B1.

Chromatography was performed using chromatography columns purchased fromAmersham Biosciences (Sephadex G25 NAP-25 prepacked columns (Amersham17-0852-02); HiPrep 26/10 Desalting Columns, Sephadex G25 fine resin, 3connected in series (Amersham 17-5087-01)). TSK-GEL G3000SWXLchromatography columns (TOSOH Bioscience, 08541) were also used, withTSK Column Guard SWxl (TOSOH Bioscience 08543).

Solvents used in the following experiments were dimethylsulfoxide(DMSO), dimethylacetamide (DMA), ethanol (EtOH), and 100 mM Ellman'sReagent (DTNB, available from Cayman Chemical) in DMSO.

Example 1A Preparation of huC242-SMCC-DM1 Conjugate

a. Preparation and Measurement of huC242 Antibody

The concentration of antibody was measured using an extinctioncoefficient of 1.48 (mg/mL)⁻¹ at 280 nm and a molecular weight of147,000 g/mole.

b. Preparation and Measurement of SMCC Stock Solution

A 20 mM solution of SMCC (6.69 mg/mL) was prepared in dimethylsulfoxide(DMSO). The solution was diluted 1/40 in Assay Buffer and the absorbanceof the samples measured at 302 nm. The concentration of the stocksolution was calculated using an extinction coefficient of 602 M⁻¹cm⁻¹.

c. Preparation and Measurement of DM1 Stock Solution

A 10 mM solution of DM1 (free thiol form) was prepared indimethylacetamide (DMA) (7.37 mg/mL) (FIG. 2). The absorbance ofdilutions of the stock solution in ethanol (EtOH) was measured at 280nm. The concentration of stock DM1 was calculated by using an extinctioncoefficient of 5700 M⁻¹ at 280 nm. The concentration of free sulfhydrylor thiol groups (—SH) in the stock DM1 preparation was measured usingEllman's reagent (DTNB). Dilutions of the stock solution were preparedin Assay buffer made to 3% (v/v) DMA, and then 100 mM DTNB in DMSO(1/100th volume) was added. The increase in absorbance at 412 nm wasmeasured against a reagent blank and the concentration was calculatedusing an extinction coefficient of 14150 M⁻¹cm⁻¹. The concentration of—SH resulting from the Ellman's assay was used to represent the DM1stock concentration in calculations for conjugation conditions.

d. Modification of huC242 with SMCC Crosslinker

The antibody was split into two samples; one was modified using a7.5-fold molar excess of SMCC cross-linker, the other with a 8.5-foldmolar excess of SMCC cross-linker. Samples were reacted at 8 mg/mLantibody. The reactions were carried out in Buffer A (95% v/v) with DMSO(5% v/v) for 2 hours at room temperature with stirring.

e. G25 Chromatography to Remove Excess SMCC

The huC242-SMCC reaction mixtures were gel-filtered through 1.5×4.9 cmpre-packed columns of Sephadex G25 resin equilibrated in Buffer A. Theload and elution volumes were according to manufacturer's instructions.The modified antibody elutions were assayed to determine theconcentration of the antibody using the extinction co-efficientdescribed above. The yield of modified antibody was 74.6% for the7.5-fold molar excess SMCC reaction and 81.6% for the 8.5-fold molarexcess SMCC reaction.

f. Conjugation of huC242-SMCC with DM1

The modified antibody samples were reacted with a 1.7-fold excess of DM1over linker (assuming 5 linkers per antibody). The reactions werecarried out at 2.5 mg/mL antibody concentration in Buffer A (97% v/v)with DMA (3% v/v). After addition of DM1, the reactions were incubatedat room temperature for approximately 20 hours with stirring.

g. Conjugation Purification by G25 Chromatography

The conjugation reaction mixtures were gel-filtered through 1.5×4.9 cmpre-packed columns of Sephadex G25 resin equilibrated in Buffer B. Theload and elution volumes were according to manufacturer's instructions.The number of DM1 molecules linked per mole of huC242 was determined bymeasuring absorbance of the eluted material at both 252 nm and 280 nm.The DM1/antibody ratio for the 7.5-fold molar excess SMCC sample wasfound to be 3.54 and the ratio for the 8.5-fold molar excess SMCC samplewas found to be 3.65. The conjugation step yields were 83.7% and 75.4%,respectively. Both conjugates were pooled together, sterile-filtered,and re-assayed for drug and antibody concentrations. The pooled samplewas assigned Lot #1713-146C and analyzed for binding, cytotoxicity,specificity, extent of aggregation and free drug content.

TABLE I Characteristics of huC242-SMCC-DM1 Final Protein Final DM1 Conc.Reference Number Conc. (mg/ml) (ug/ml) DM1/Ab 1713-146C 1.77 26.96 3.05

Example 1B In Vitro Testing of huC242-SMCC-DM1

a. Binding

The binding affinities of huC242 antibody and huC242-SMCC-DM1 werecompared using an indirect method on COLO 205 cells, where 5×10³ cellsper well were used, with three hour primary incubation on ice. Theresults are shown in FIG. 3. The naked antibody bound with a KD of5.1×10⁻¹⁰ M and the conjugated version bound with a KD of 5.52×10⁻¹⁰ M.Thus, conjugation with DM1 does not appear to alter the binding affinityof huC242.

b. Cytotoxicity and Specificity

The in vitro cytotoxicity and specificity of the huC242-SMCC-DM1conjugate were evaluated using a continuous exposure clonogenic assay.The results are shown in FIG. 4. HuC242-SMCC-DM1 was effective indestroying the antigen-positive SKBR3 cells (IC₅₀=3.5×10⁻¹² M).Specificity was shown by comparing the IC₅₀ value of the target SKBR3cells to that of the antigen-negative cell line, A375, in which the IC₅₀of the conjugate was greater than 3.0×10⁻⁹ M.

c. Size Exclusion Chromatography Analysis

The conjugate was analyzed using a TSK3000 size exclusion column (FIG.5). Peak 4 represents the monomer fraction of the conjugate, whileearlier peaks represent multimer and later peaks represent fragment. Thearea under each curve divided by the total peak areas represents thepeak's contribution to the sample. The conjugate sample was found to be96.0% monomer.

d. Free Drug

The percent of free drug was measured by ELISA and was found to be 4.4%.

Example 2A Preparation of Trastuzumab-SMCC-DM1 Conjugate

Trastuzumab antibody was obtained from Genentech for conjugation to DM1using the non-cleavable heterobifunctional cross-linking reagent SMCC.The antibody was buffer-exchanged from 50 mM potassium phosphate/2 mMEDTA, pH 6.0 into 50 mM potassium phosphate/50 mM sodium chloride/2 mMEDTA, pH 6.5 (Buffer A). The antibody was then reacted with 7.5-foldmolar excess SMCC linker and purified by Sephadex G25 resin before itwas conjugated with DM1. The final conjugate was again purified bySephadex G25 resin. The resulting conjugate contained 3.1 moles of DM1per mole of antibody.

a. Preparation and Measurement of Trastuzumab Antibody

Trastuzumab antibody in 50 mM potassium phosphate/2 mM EDTA, pH 6.0buffer was passed over a Sephadex G25 column equilibrated with Buffer Aand eluted into Buffer A. All buffers used in this experiment weretested to be free of endotoxin using a chromogenic Lymulus amoebocytelysate (LAL) method (Cambrex). The concentration of antibody wasmeasured using an extinction coefficient of 1.45 mL mg⁻¹ cm⁻¹ at 280 nmand a molecular weight of 145,423 g.

b. Preparation and Measurement of SMCC Stock Solution

A 20 mM solution of SMCC (6.69 mg/mL) was prepared in DMSO. The solutionwas diluted 1/40 in Assay Buffer and the absorbance of the samples wasmeasured at 302 nm. The concentration of the stock solution wascalculated using a molar extinction coefficient of 602 M⁻¹cm⁻¹.

c. Preparation and Measurement of DM1 Stock Solution

A 10 mM solution of DM1 (free thiol form) was prepared in DMA (7.37mg/mL) (FIG. 2). The absorbance of dilutions of the stock solution inEtOH was measured at 280 nm. The concentration of stock DM1 wascalculated by using a molar extinction coefficient of 5700 M⁻¹cm⁻¹ at280 nm. The concentration of free —SH in the stock DM1 preparation wasmeasured using Ellman's reagent (DTNB). Dilutions of the stock solutionwere prepared in Assay buffer made to 3% (v/v) DMA, and then 100 mM DTNBin DMSO (1/100^(th) volume) was added. The increase in absorbance at 412nm was measured against a reagent blank and the concentration wascalculated using an extinction coefficient of 14150 M⁻¹cm⁻¹. Theconcentration of —SH resulting from the Ellman's assay was used torepresent the DM1 stock concentration in calculations for conjugationconditions.

d. Modification of Trastuzumab with SMCC Crosslinker

The antibody was modified using a 7.5-fold molar excess of SMCC at 20mg/mL antibody. The reaction was carried out in Buffer A (95% v/v) withDMSO (5% v/v) for 2 hours at room temperature with stirring.

e. G25 Chromatography to Remove Excess SMCC

The trastuzumab-SMCC reaction mixture was gel-filtered through a 1.5×4.9cm pre-packed column of Sephadex G25 resin equilibrated in Buffer A. Theload and elution volumes were according to manufacturer's instructions(Amersham Biosciences). The concentration of the modified antibodysolution was assayed spectrophotometrically using the extinctionco-efficient described above. The yield of modified antibody was 88%based on protein concentration.

f. Conjugation of Trastuzumab-SMCC with DM1

The modified antibody was reacted with a 1.7-fold excess of DM1 overlinker (assuming 5 linkers per antibody). The reaction was carried outat 10 mg/mL antibody concentration in Buffer A (94% v/v) with DMA (6%v/v). After addition of DM1, the reaction was incubated at roomtemperature for 16.5 hours with stirring.

g. Conjugation Purification by G25 Chromatography

The conjugation reaction mixture was gel-filtered through a 1.5×4.9 cmpre-packed column of Sephadex G25 resin equilibrated in Buffer B. Theload and elution volumes were according to manufacturer's instructions(Amersham Biosciences). The number of DM1 molecules linked per mole oftrastuzumab was determined by measuring absorbance at both 252 nm and280 nm of the eluted material. The DM1/antibody ratio was found to be3.13 and the conjugation step yield was 95.7%. The overall yield ofconjugated trastuzumab was 84% based on the starting antibody. Theresulting conjugate was analyzed for binding, cytotoxicity, specificity,extent of aggregation and free drug content.

TABLE II Characteristics of Trastuzumab-SMCC-DM1 Final Protein Final DM1Conc. Reference Number Conc. (mg/ml) (ug/ml) DM1/Ab 1762-14 6.71 106.3.13

Example 2B In Vitro Testing of Trastuzumab-SMCC-DM1

Binding studies showed that the conjugation of antibody to DM1 did notaffect the apparent K_(D); both naked trastuzumab antibody andtrastuzumab-SMCC-DM1 conjugate had the same binding affinity to ECDplates (5.5×10⁻¹¹ M). Evaluation of the in vitro cytotoxicity of thesample showed that the trastuzumab-SMCC-DM1 conjugate is both highlytoxic (IC₅₀ 3.6×10⁻¹² M on antigen-positive cell line) and specific(IC₅₀ greater than 3.0×10⁻⁹ M on antigen-negative cell line).

a. Binding

The binding affinity of trastuzumab antibody and trastuzumab-SMCC-DM1were compared using the HER2 ECD plate-binding assay provided byGenentech. The results are shown in FIG. 24. Both the naked antibody andconjugated version bind with an apparent K_(D) of 5.5×10⁻¹¹ M. Thus,conjugation with DM1 does not alter the binding affinity of trastuzumab.

b. Cytotoxicity and Specificity

The in vitro cytotoxicity and specificity of the trastuzumab-SMCC-DM1conjugate were evaluated using a continuous exposure clonogenic assay.The results are shown in FIG. 25. Trastuzumab-SMCC-DM1 was effective indestroying the antigen-positive SKBR3 cells (IC₅₀=3.6×10⁻¹² M).Specificity was shown when comparing the IC₅₀ of the target SKBR3 cellsto the antigen-negative cell line, A375, in which the IC₅₀ of theconjugate was greater than 3.0×10⁻⁹ M.

c. Size Exclusion Chromatography Analysis

The conjugate was analyzed using a TSK3000 size exclusion column (FIG.26). Peak 1 represents multimer, peak 2 represents dimer, and peak 3represents monomer. The area under each curve divided by total peakareas represents the peak's contribution to the sample. The conjugatesample was found to be 95.3% monomer.

d. Free Drug Analysis

The percent free drug was measured by ELISA and found to be 3.4%.

e. Endotoxin Level

The conjugate was tested using a chromatographic LAL test and found tocontain 0.03 EU/mg.

Example 3A Preparation of Trastuzumab-SIAB-DM1 Conjugate

Trastuzumab antibody was obtained from Genentech for conjugation to DM1using the non-cleavable heterobifunctional crosslinker SIAB. Theantibody was reacted with 7.0-fold molar excess SIAB linker at pH 6.5and purified by Sephadex G25F resin. Antibody containing fractions werepooled and reacted with DM1 overnight at standard conjugation conditionsof pH 6.5 and room temperature but in the dark. An aliquot was removedfrom the reaction vessel and analyzed to determine incorporation of DM1.The aliquot was measured after a NAP 5 filtration to have only 1.4drugs/Ab. An additional 8-fold excess of SIAB was added to the reactionfor 2 hours and then the pH was increased to 8 just prior to theaddition of an additional 1.5-fold excess DM1/SIAB. The reaction wasallowed to proceed and was purified using Sepahadex G25F resin. Theresulting conjugate contained 3.42 moles of DM1 per mole of antibody.

a. Measurement of Trastuzumab Antibody

The concentration of antibody was measured using an extinctioncoefficient of 1.45 mL mg⁻¹ cm⁻¹ at 280 nm and a molecular weight of145,423 g.

b. Preparation and Measurement of SIAB Stock Solution

An 18 mM solution of SIAB (7.2 mg/mL) was prepared in DMSO. A wavelengthscan of the solution diluted into pH 4 buffer was recorded forinformational purposes only.

c. Preparation and Measurement of DM1 Stock Solution

An approximately 30 mM solution of DM1 (free thiol form) was prepared inDMA. The concentration of free —SH in the stock DM1 preparation wasmeasured using Ellman's reagent (DTNB). Dilutions of the stock solutionwere prepared in Assay buffer made to 3% (v/v) DMA, and then 100 mM DTNBin DMSO (1/100^(th) volume) was added. The increase in absorbance at 412nm was measured against a reagent blank and the concentration wascalculated using a molar extinction coefficient of 14150 M⁻¹cm⁻¹. Theconcentration of —SH resulting from the Ellman's assay was used torepresent the DM1 stock concentration in calculations for conjugationconditions.

d. Modification of Trastuzumab with SIAB Cross Linker

The antibody was modified using a 7.0-fold molar excess of SIAB at 20mg/mL antibody. The reaction was carried out in Buffer A (95% v/v) withDMSO (5% v/v) for 2 hours at room temperature with stirring in the dark.

e. G25 Chromatography to Remove Excess SIAB

The Trastuzumab-SIAB reaction mixture was gel-filtered through HiPrep26/10 Desalting Columns equilibrated in Buffer A. There appeared to beinterference at 280 nm from the SIAB reagent, so the yield of modifiedantibody was assumed to be 100% and a modification of 5 linkers/antibodywas assumed for determination of the amount of DM1 in the conjugationreaction.

f. Conjugation of Trastuzumab-SIAB with DM1

The modified antibody was reacted with a 1.7-fold excess of DM1 overlinker assuming 100% yield and 5 cross-linkers/antibody as stated above.The concentration of antibody in the reaction was estimated to be 12.5mg/mL and the reaction was carried out in Buffer A (97% v/v) with DMA(3% v/v). After addition of DM1, the reaction was incubated at roomtemperature in the dark for 16.5 hours with stirring.

g. Conjugation Reaction Analysis

A 0.25 mL aliquot of the reaction mixture was removed and gel-filteredover a prepacked G25 Sephadex column equilibrated in Buffer B. Thenumber of DM1 molecules linked per mole of trastuzumab was determined bymeasuring absorbance at both 252 nm and 280 nm of the eluted material.The DM1/antibody ratio was only 1.4.

h. Additional Modification/Conjugation Reaction

An additional 8-fold molar excess of SIAB was added and allowed toincubate for 2 hours at room temperature. A 1.5 fold molar excess of DM1over SIAB was added and the pH of the reaction was increased to 8 withthe addition of 1 N NaOH. The reaction was incubated at room temperaturein the dark and gel-filtered over a column of G25F resin equilibratedinto Buffer B.

i. Pooling and Characterization of Conjugate

Protein containing fractions were pooled, filtered and measured byabsorbance at 252 and 280 nm. Samples of the conjugate were tested forendotoxin level, binding, specific and non-specific cytotoxicity, %monomer and free drug level.

TABLE III Characteristics of Trastuzumab-SIAB-DM1 Final Protein FinalDM1 Conc. Reference Number Conc. (mg/ml) (ug/ml) DM1/Ab 1806-32 5.6297.3 3.42

Example 3B In Vitro Testing of Trastuzumab-SIAB-DM1

Binding studies showed that the conjugation of antibody to DM1 did notaffect the apparent K_(D); both naked trastuzumab andtrastuzumab-SIAB-DM1 had a similar binding affinities (1.2×10⁻¹⁰M Ab and1.9×10⁻¹⁰M apparent K_(D) conjugate). Evaluation of the in vitrocytotoxicity of the sample showed that the trastuzumab-SIAB-DM1conjugate is both highly toxic (IC₅₀ 5×10⁻¹²M on antigen-positive cellline SKBR3) and specific (IC₅₀ greater than 3.0×10⁻⁹M onantigen-negative cell line, A375).

a. Binding

The binding affinity of trastuzumab antibody and trastuzumab-SIAB-DM1were compared using the HER2 ECD plate binding assay provided byGenentech. The results are shown in FIG. 27. Naked trastuzumab andtrastuzumab-SIAB-DM1 had similar binding affinities (1.2×10⁻¹⁰ M for theantibody and 1.9×10⁻¹⁰ M apparent K_(D) for the conjugate).

b. Cytotoxicity and Specificity

Evaluation of the in vitro cytotoxicity of the sample showed that thetrastuzumab-SIAB-DM1 conjugate is both highly toxic (IC₅₀₌5×10⁻¹² M onantigen-positive cell line, SKBR3) and specific (IC₅₀ greater than3.0×10⁻⁹ M on antigen-negative cell line, A375). See FIG. 28.

c. Size Exclusion Chromatography Analysis

The conjugate was analyzed using a TSK3000 size exclusion column (FIG.29). Peak 1 represents dimer and peak 2 represents monomer. The areaunder each curve divided by total peak areas represents the peak'scontribution to the sample. The conjugate sample was found to be 96.4%monomer.

d. Free Drug

The percent of free drug was measured by ELISA and was found to be0.35%.

e. Endotoxin Level

The conjugate was tested using a chromatographic LAL test and found tocontain <0.04 EU/mg.

Example 4 Conjugation of huC242 with a Cross-Linking Reagent that Formsa Non-S-Containing Non-Cleavable Linker

a. Synthesis

A stock solution of the cross-linking reagent (see FIG. 21 forstructure) was made up in DMA, insoluble precipitate was spun out, andthe concentration of the remaining solution was determined using anextinction coefficient of ε²⁸⁰=5700 M⁻¹ cm⁻¹ which is the extinction forDM1 at this wavelength. Since the real extinction coefficient for thismaterial has not been measured this is only an estimate ofconcentration. It should be noted that the ratio ε²⁵²/ε²⁸⁰ for DM1 is4.7 (in ETOH) while ε²⁵²/ε²⁸⁰ for this cross-linking reagent solution(in pH 7.5 buffer) was measured as 1.42 suggesting either differentextinctions or impurities.

The conjugation reaction was carried out on a 2 mg scale using 2.8 mg/mlhuC242 antibody in 16% DMA in Buffer E, pH 7.5 (Buffer E=50 mM sodiumphosphate, 150 mM NaCl, 10 mM EDTA). Based on the estimatedcross-linking reagent concentration of the stock solution, 30equivalents of cross-linker/antibody were used (an earlier experimentusing 10 eq. of cross-linker/antibody produced a conjugate with only 0.9DM1/antibody). The reaction was allowed to go for 3 hours and then theconjugate was purified by passage over a Nap 10 (G25) column. Afterfiltering (Millex GV filter, 0.2 um pore size), the conjugate had 2.56DM1/antibody (Lot #1749-119A, antibody recovery=78%). An aliquot of theconjugate was examined by HPLC (HiPrep column) for free DM1 and asizable DM1 peak was observed at 12.09′. The sample was thereforedialyzed in Buffer B to get rid of this peak and then reassayed. Thefinal conjugate sample (Lot #1749-124A) had no free DM1 by HPLC and had1.84 DM1/antibody. SEC HPLC was carried out on the conjugate to showthat it was 97% monomeric antibody.

b. Cytotoxicity and Binding

The inventors carried out binding and cytotoxicity studies on thehuC242-non-S-containing non-cleavable linker-DM1 conjugate. First, thebinding affinities of huC242 antibody, huC242-SMNP-DM3, andhuC242-non-S-containing non-cleavable linker-DM1 to COLO 205 cells werecompared. 5×10³ cells per well were used, with a three hour primaryincubation on ice. The results are shown in FIG. 23. Thehu242-non-S-containing non-cleavable linker-DM1 conjugate had about atwo-fold higher apparent dissociation constant than free antibody (seeFIG. 23). In addition the huC242-non-S-containing non-cleavablelinker-DM1 conjugate had an in vitro cytotoxicity comparable tohuC242-SMNP-DM3 (IC₅₀ of the non-S-containing non-cleavable linkerconjugate=7.0×10⁻¹² M) (see FIG. 22).

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope of the invention.

All patents, publications, and other references cited herein areexpressly incorporated by reference in their entireties.

What is claimed is:
 1. A cell-binding agent maytansinoid conjugatehaving at least one maytansinoid linked to a cell-binding agent via anon-cleavable linker, wherein the maytansinoid-linker portion of theconjugate is represented by the following formulae:

wherein X is a linear or branched alkyl, alkenyl or alkynyl groupbearing 2 to 20 carbon atoms, Y is a cycloalkyl or cycloalkenyl groupbearing 3 to 10 carbon atoms, Z is a substituted or unsubstitutedaromatic group bearing 6 to 10 carbon atoms or a substituted orunsubstituted heterocyclic group wherein the hetero atom is selectedfrom N, O or S, and l, m and n are each 0 or 1, provided that they areall not 0 at the same time,

represents a bond to the cell binding agent; and May represents amaytansinoid that bears a side chain at C-3 hydroxyl, C-14hydroxymethyl, C-15 hydroxyl or C-20 desmethyl.
 2. The cell bindingagent maytansinoid conjugate of claim 1, wherein an average of 1 toabout 10 maytansinoids is covalently linked to the cell-binding agent.3. The cell-binding agent maytansinoid conjugate of claim 1, wherein themaytansinoid-linker portion of the conjugate is represented by thefollowing formula:

wherein n′ represents an integer from 3 to
 24. 4. The cell binding agentmaytansinoid conjugate of claim 3, wherein an average of 1 to about 10maytansinoids is covalently linked to the cell-binding agent.
 5. Thecell-binding agent maytansinoid conjugate of claim 1, wherein themaytansinoid-linker portion of the conjugate is represented by thefollowing formula:


6. The cell binding agent maytansinoid conjugate of claim 5, wherein anaverage of about 1 to 10 maytansinoids is covalently linked to thecell-binding agent.
 7. The cell binding agent maytansinoid conjugate ofclaim 3, wherein the maytansinoid-linker portion of the conjugate isrepresented by the following formula:


8. The cell binding agent maytansinoid conjugate of claim 7, wherein anaverage of about 1 to 10 maytansinoids is covalently linked to thecell-binding agent.
 9. The cell-binding agent maytansinoid conjugate ofclaim 1, wherein the cell-binding agent binds to tumor cells; virusinfected cells, microorganism infected cells, parasite infected cells,autoimmune cells, activated cells in graft vs. host disease, myeloidcells, activated T-cells, B cells, or melanocytes; cells expressing theCD33, CD19, CanAg, CALLA, or Her-2 antigens; or cells expressing insulingrowth factor receptor, epidermal growth factor receptor, prostatespecific membrane antigen (PSMA) or folate receptor.
 10. Thecell-binding agent maytansinoid conjugate of claim 1, wherein thecell-binding agent binds to kidney cancer cells, lung cancer cells,breast cancer cells, prostate cancer cells, ovarian cancer cells,colorectal cancer cells, gastric cancer cells, squamous cancer cells,small-cell lung cancer cells, non-small-cell lung cancer cells,pancreatic cancer cells, testicular cancer cells, neuroblastoma cells,melanoma cells or cells from cancer of the lymphatic organs.
 11. Thecell-binding agent maytansinoid conjugate of claim 1, wherein thecell-binding agent is an antibody, a single chain antibody, an antibodyfragment that specifically binds to a target cell, a monoclonalantibody, a single chain monoclonal antibody, or a monoclonal antibodyfragment that specifically binds to a target cell, a chimeric antibody,a chimeric antibody fragment that specifically binds to a target cell, adomain antibody, a domain antibody fragment that specifically binds to atarget cell, a lymphokine, a hormone, a vitamin, a growth factor, acolony stimulating factor, or a nutrient-transport molecule.
 12. Thecell-binding agent maytansinoid conjugate of claim 1, wherein thecell-binding agent is an interferon, IL2, IL3, IL4, IL6, insulin,thyrotropin releasing hormone, melanocyte-stimulating hormone, a steroidhormone, somatostatin, EGF, TGF-α, FGF, G-CSF, VEGF, MCSF, GM-CSF, folicacid, transferrin, estrogen, estrogen analogues, androgen, or androgenanalogues.
 13. The cell-binding agent maytansinoid conjugate of claim 1,wherein the cell-binding agent is a resurfaced antibody, a resurfacedsingle chain antibody, or a resurfaced antibody fragment thatspecifically bind to a target cell.
 14. The cell-binding agentmaytansinoid conjugate of claim 1, wherein the cell-binding agent is ahumanized antibody, a humanized single chain antibody, or a humanizedantibody fragment that specifically bind to a target cell.
 15. Thecell-binding agent maytansinoid conjugate of claim 1, wherein thecell-binding agent is a human antibody, a human single chain antibody,or a human antibody fragment that specifically bind to a target cell.16. The cell-binding agent maytansinoid conjugate of claim 1, whereinthe cell-binding agent is a resurfaced monoclonal antibody, a resurfacedsingle chain monoclonal antibody, or a resurfaced monoclonal antibodyfragment that specifically bind to tumor cells.
 17. The cell-bindingagent maytansinoid conjugate of claim 1, wherein the cell-binding agentis a humanized monoclonal antibody, a humanized single chain monoclonalantibody, or a humanized monoclonal antibody fragment that specificallybind to tumor cells.
 18. The cell-binding agent maytansinoid conjugateof claim 1, wherein the cell-binding agent is a human monoclonalantibody, a human single chain monoclonal antibody, or a humanmonoclonal antibody fragment that specifically bind to tumor cells. 19.The cell-binding agent maytansinoid conjugate of claim 1, wherein thecell-binding agent is a resurfaced monoclonal antibody, a resurfacedsingle chain monoclonal antibody, or a resurfaced monoclonal antibodyfragment that specifically bind to colorectal cancer cells, breastcancer cells, kidney cancer cells, lung cancer cells, prostate cancercells, ovarian cancer cells, gastric cancer cells, squamous cancercells, small-cell lung cancer cells, non-small-cell lung cancer cells,pancreatic cancer cells, testicular cancer cells, neuroblastoma cells,melanoma cells or cells from cancer of the lymphatic organs.
 20. Thecell-binding agent maytansinoid conjugate of claim 1, wherein thecell-binding agent is a humanized monoclonal antibody, a humanizedsingle chain monoclonal antibody, or a humanized monoclonal antibodyfragment that specifically bind to colorectal cancer cells, breastcancer cells, kidney cancer cells, lung cancer cells, prostate cancercells, ovarian cancer cells, gastric cancer cells, squamous cancercells, small-cell lung cancer cells, non-small-cell lung cancer cells,pancreatic cancer cells, testicular cancer cells, neuroblastoma cells,melanoma cells or cells from cancer of the lymphatic organs.
 21. Thecell-binding agent maytansinoid conjugate of claim 1, wherein thecell-binding agent is a human monoclonal antibody, a human single chainmonoclonal antibody, or a human monoclonal antibody fragment thatspecifically bind to colorectal cancer cells, breast cancer cells,kidney cancer cells, lung cancer cells, prostate cancer cells, ovariancancer cells, gastric cancer cells, squamous cancer cells, small-celllung cancer cells, non-small-cell lung cancer cells, pancreatic cancercells, testicular cancer cells, neuroblastoma cells, melanoma cells orcells from cancer of the lymphatic organs.
 22. The cell-binding agentmaytansinoid conjugate of claim 1, wherein the cell-binding agent is aresurfaced monoclonal antibody, a resurfaced single chain monoclonalantibody, or a resurfaced monoclonal antibody fragment that specificallybind to breast cancer cells, colorectal cancer cells, pancreatic cancercells, non-small cell lung cancer cells and gastric cancer cells. 23.The cell-binding agent maytansinoid conjugate of claim 1, wherein thecell-binding agent is a humanized monoclonal antibody, a humanizedsingle chain monoclonal antibody, or a humanized monoclonal antibodyfragment that specifically bind to breast cancer cells, colorectalcancer cells, pancreatic cancer cells, non-small cell lung cancer cellsand gastric cancer cells.
 24. The cell-binding agent maytansinoidconjugate of claim 1, wherein the cell-binding agent is a humanmonoclonal antibody, a human single chain monoclonal antibody, or ahuman monoclonal antibody fragment that specifically bind to breastcancer cells, colorectal cancer cells, pancreatic cancer cells,non-small cell lung cancer cells and gastric cancer cells.
 25. Thecell-binding agent maytansinoid conjugate of claim 1, wherein thecell-binding agent is an anti-PSMA antibody, an anti-CanAg antibody, ananti-CD19 antibody, an anti-CD33 antibody, an anti-CALLA antibody, ananti-EGFR antibody, an anti-CD56 antibody, or an anti-IGF-IR antibody,or an anti-Her2 antibody.
 26. The cell-binding agent maytansinoidconjugate of claim 1, wherein the cell-binding agent is an anti-erbBantibody, a single chain anti-erbB antibody or an anti-erbB antibodyfragment that specifically bind to a target cell, wherein at least aconstant region of the antibody, single chain antibody or antibodyfragment comprises a human antibody amino acid sequence.
 27. Thecell-binding agent maytansinoid conjugate of claim 1, wherein thecell-binding agent is trastuzumab.
 28. The cell-binding agentmaytansinoid conjugate of claim 1, wherein the cell-binding agent is anantibody fragment of trastuzumab selected from Fab, Fab′, F(ab′)₂, andFv fragments of trastuzumab.
 29. The cell-binding agent maytansinoidconjugate of claim 1, wherein the cell-binding agent is a resurfaced orhumanized My9-6 or N901 antibody or fragment thereof, wherein said My9-6antibody comprises a heavy chain and a light chain, said heavy chaincomprising three complementarity determining regions comprising HCCDR1,HCCDR2 and HCCDR3 of murine antibody My9-6, and wherein said light chaincomprises three complementarity determining regions comprising LCCDR1,LCCDR2 and LCCDR3 of murine antibody My9-6; and wherein said resurfacedor humanized N901 antibody or fragment thereof comprises a heavy chainand a light chain, said heavy chain comprising three complementaritydetermining regions comprising HCCDR1, HCCDR2 and HCCDR3 of murineantibody N901, and said light chain comprising three complementaritydetermining regions comprising LCCDR1, LCCDR2 and LCCDR3 of murineantibody N901.
 30. The cell-binding agent maytansinoid conjugate ofclaim 1, wherein the cell-binding agent is a resurfaced or humanized B4antibody, wherein said resurfaced or humanized B4 antibody comprises aheavy chain and a light chain, said heavy chain comprising threecomplementarity determining regions comprising HCCDR1, HCCDR2 and HCCDR3of murine antibody B4, and said light chain comprising threecomplementarity determining regions comprising LCCDR1, LCCDR2 and LCCDR3of murine antibody B4.
 31. The cell-binding agent maytansinoid conjugateof claim 1, wherein the cell-binding agent is a resurfaced or humanizedC242 antibody or fragment thereof, wherein said resurfaced or humanizedC242 antibody comprises a heavy chain and a light chain, said heavychain comprising three complementarity determining regions comprisingHCCDR1, HCCDR2 and HCCDR3 of murine antibody C242, and said light chaincomprising three complementarity determining regions comprising LCCDR1,LCCDR2 and LCCDR3 of murine antibody C242.
 32. A composition comprisingthe cell-binding agent maytansinoid conjugate of claim 1 and a carrier.33. A process of making the cell-binding agent maytansinoid conjugate ofclaim 1, the process comprising: (a) providing the maytansinoid, (b)modifying the maytansinoid with a cross-linking agent to yield amaytansinoid ester, and (c) conjugating the maytansinoid ester with thecell-binding agent, thereby providing the non-cleavable linker betweenthe cell-binding agent and the maytansinoid to produce the conjugate.34. A method for targeting maytansinoids to a selected cell population,the method comprising contacting a cell population or tissue suspectedof containing the selected cell population with the cell-binding agentmaytansinoid conjugate of claim
 1. 35. A method of eliminating cells,the method comprising contacting the cells with the cell-binding agentmaytansinoid conjugate of claim
 1. 36. A method for treatment of tumors,the method comprising administering to a subject in need of treatment aneffective amount of the cell-binding agent maytansinoid conjugate ofclaim
 1. 37. The cell binding agent maytansinoid conjugate of claim 1,wherein the maytansinoid-linker portion of the conjugate is representedby the following formula: